1. Field of the Invention
The present invention is generally in the field of modulators of apoptopic cell death and uses thereof in therapeutic applications to inhibit or to enhance apoptosis, as desired depending on the disease and whether or not it is desired to kill the diseased cells or to rescue the diseased cells from apoptopic cell death. Specifically, the present invention concerns novel genes encoding novel proteins belonging to the leucine zipper family, which are capable of inhibiting apoptosis mediated by the CD3/TCR system or by the Fas/Fas-L system, and which are also capable of stimulating lymphocyte activation. In particular, the present invention concerns a new protein and the gene encoding therefor called GILR, preparation and uses thereof, as well as any isoforms, analogs, fragments and derivatives of GILR, their preparation and uses.
2. Description of the Related Art
Apoptosis (programmed cell death) is an important intracellular process having an important role in normal cell and tissue development as well as in the control of neoplastic growth (Cohen, 1993; Osborne and Schwartz, 1994; Wyllie et al., 1980; Kerr et al., 1972; Bursch et al., 1992).
A number of stimuli can either induce or inhibit programmed cell death through activation of molecules, involved in the signaling and execution of apoptosis, acting at different levels including the cell membrane, cytoplasm and nucleus. Among these, of note are those intracellular molecules, including some transcription factors, that have been shown to regulate cell growth. In particular, leucine zipper family proteins, such as for instance MYC, FOS and JUN, can modulate cell death (Shi et al., 1992; Smeyne et al., 1993; Goldstone and Lavin, 1994).
Apoptosis is also important in T-cell development (Dent et al., 1990; Ju et al., 1995; MacDonald and Lees, 1990). In particular, negative selection is due to apoptosis activated through the antigen (Ag) interaction with the T-cell-receptor(TCR)/CD3 complex (Smith et al., 1989). Engagement of the TCR/CD3 complex, either by APCs presenting antigenic peptide or by anti-CD3 antibody, triggers a series of activation events, such as for example, the expression of the Fas/Fas-Ligand (Fas/Fas-L) system, that can induce apoptosis in thymocytes, mature T cells and T cell hybridoma (Alderson et al., 1995; Dhein et al., 1995; Ju et al., 1995; Jenkinson et al., 1989; Webb et al., 1990; Yang et al., 1995). For example, triggering of such activation events in T cell hybridomas leads to cell cycle arrest, followed by apoptosis. This activation-induced cell death (AICD, Kabelitz et al., 1993) requires the interaction of Fas with Fas-L (Alderson et al., 1995; Itoh et al., 1991; Yang et al., 1995).
It has been shown that other stimuli, such as cytokines and glucocorticoid hormones (GCH), are also critical regulators of T-cell development (Migliorati et al., 1993; Nieto et al., 1990; Nieto and Lopez-Rivas, 1989; Cohen and Duke, 1984; Wyllie, 1980). For example, dexamethasone (DEX), a synthetic GCH which by itself induces apoptosis in T cell hybridomas and in normal T lymphocytes, can inhibit AICD induced by triggering of the TCR/CD3 complex (Zacharchuk et al., 1990). This inhibition may be due to prevention of activation induced expression of Fas and Fas-L (Yang et al., 1995).
With respect to the above noted Fas/Fas-L system, it should be noted that Fas has also been called the FAS receptor or FAS-R as well as CD95. For simplicity, this receptor will be called ‘Fas’ herein throughout and its ligand, as noted above, will be called ‘Fas-L’ herein throughout.
Fas is member of the TNF/NGF superfamily of receptors and it shares homology with a number of cell-surface receptors including the p55-TNF receptor and the NGF receptor (see for example Boldin et al., 1995a and 1995b). Fas mediates cell death by apoptosis (Itoh et al. 1991) and appears to act as a negative selector of autoreactive T cells, i.e. during maturation of T-cells, Fas mediates the apoptopic death of T cells recognizing self-antigens. Mutations in the Fas gene, such as the lpr mutations in mice, have been shown to be responsible for a lymphoproliferation disorder in mice resembling the human autoimmune disease, systemic lupus erythomatosus (SLE; Watanabe-Fukunaga et al., 1992). The Fas-L molecule is apparently a cell surface-associated molecule carried by, amongst others, killer T cells (or cytotoxic T lymphocytes—CTLs), and hence, when such CTLs contact cells carrying Fas, they are capable of inducing apoptopic cell death of the Fas-carrying cells. Further, a monoclonal antibody specific to Fas has been prepared which is capable of inducing apoptopic cell death in cells carrying Fas, including mouse cells transformed by cDNA encoding human Fas (see, for example, Itoh et al,. 1991).
While some of the cytotoxic effects of lymphocytes are mediated by interaction of lymphocyte-produced Fas-L with the widely-occurring Fas, it has also been found that various other normal cells besides T lymphocytes, express Fas on their surface and can be killed by the triggering of this receptor. Uncontrolled induction of such a killing process is suspected to contribute to tissue damage in certain diseases, for example, the destruction of liver cells in acute hepatitis. Accordingly, finding ways to restrain the cytotoxic activity of Fas may have therapeutic potential.
Conversely, since it has also been found that certain malignant cells and HIV-infected cells carry Fas on their surface, antibodies against Fas, or Fas-L itself, may be used to trigger Fas-mediated cytotoxic effects in these cells and thereby provide a means for combating such malignant cells or HIV-infected cells (see, for example, Itoh et al. 1991). Finding yet other ways for enhancing the cytotoxic activity of Fas may therefore also have therapeutic potential.
As noted above, Fas is related to one of the TNF receptors, namely, the p55-TNF receptor. TNF (both TNF-α and TNF-β, and as used throughout, ‘TNF’ will refer to both) has many effects on cells (see, for example, Wallach, D. (1986) In: Interferon 7 (Ion Gresser ed.), p. 83-122, Academic Press, London; and Beutler and Cerami (1987)). TNF exerts its effects by binding to its receptors, the p55-TNF receptor and the p75-TNF receptor. Some of the TNF-induced effects are beneficial to the organism, such as, for example, destruction of tumor cells and virus-infected cells, and augmentation of antibacterial activities of granulocytes. In this way TNF contributes to the defense of the organism against tumors and infectious agents and contributes to recovery from injury. Thus, TNF can be used as an anti-tumor and anti-infectious agent.
However, TNF can also have deleterious effects. For example, overproduction of TNF can have a pathogenic role in several diseases, including amongst others, septic shock (Tracey et al., 1986); excessive weight loss (cachexia); tissue damage in rheumatic diseases (Beutler and Cerami, 1987); tissue damage in graft-versus-host reactions (Piquet et al., 1987); and tissue damage in inflammation, to name but a few of the pathogenic effects of TNF.
The above cytocidal effects of TNF is mediated mainly by the p55-TNF receptor in most cells studied so far, which activity is dependent on the integrity of the intracellular domain of this receptor (see, for example, Brakebusch et al., 1992; Tartaglia et al., 1993). In addition, mutational studies indicate that the related Fas and p55 TNF-receptor mediate intracellular signaling processes, ultimately resulting in cell death, via distinct regions within their intracellular domains (see also, for example, Itoh and Nagata, 1993). These regions also designated ‘death domains’ present in both these receptors, have sequence similarity. The “death domains” of Fas and p55-TNF receptor are capable of self-association, which is apparently important for promoting the receptor aggregation necessary for initiating intracellular signaling (see, for example, Song et al. 1994; Wallach et al., 1994; Boldin et al., 1995a, b), and which, at high levels of receptor expression, can result in the triggering of ligand-independent signaling (Boldin et al., 1995a, b).
Recent studies have indicated that the cytotoxic effects mediated by Fas and p55-TNF receptor involves an intracellular signaling pathway which includes a number of protein-protein interactions, leading from the initial ligand-receptor binding to the eventual activation of enzymatic effector functions, and which include non-enzymatic protein-protein interactions which are involved in the initiation of the signaling for cell death (see also, for example, Nagata and Golstein, 1995; Vandenabeele et al., 1995; and Boldin et al., 1995a, b). Apparently the binding of the trimeric Fas-L and TNF to their receptors results in the interaction of the intracellular domains of these receptors which is augmented by a propensity of the death-domain regions or motifs to self-associate (Boldin et al., 1995a, b), and induced binding of at least two other cytoplasmatic proteins (which can also bind to each other) to the intracellular domains of these receptors, namely, the protein MORT-1 (also called FADD) which binds to Fas (see Boldin et al., 1995b; Chinnaiyan et al., 1995; Kischkel et al., 1995), and the protein TRADD which binds to the p55-TNF receptor (see Hsu et al., 1995; Hsu et al., 1996). A third such intracellular protein has also been identified called RIP (see Stanger et al., 1995) which binds to the intracellular domains of both Fas and the p55-TNF receptor. RIP can also interact with TRADD and MORT-1. Thus, these three intracellular proteins allow for a functional “cross-talk” between Fas and the p55-TNF receptor. The interactions between these receptors and their associated proteins (MORT-1, TRADD, RIP) occurs through the ‘death domain’ motifs present in each of these receptors and proteins.
Thus, the “death domain” motifs of the p55-TNF receptor and Fas as well as their three associated proteins MORT-1, RIP and TRADD appear to be the sites of protein-protein interactions. The three proteins MORT-1, RIP and TRADD interact with the p55-TNF receptor and Fas intracellular domains by the binding of their death domains to those of the receptors, and for both RIP and TRADD their death domains also self-associate, although MORT-1 differs in this respect in that its death domain does not self-associate. Accordingly, it would seem that the interaction between the three proteins MORT-1, RIP and TRADD is an important part of the overall modulation of the intracellular signaling mediated by these proteins. Interference of the interaction between these three intracellular proteins will result in modulation of the effects caused by this interaction. For example, inhibition of TRADD binding to MORT-1 may modulate the Fas-p55 TNF-receptor interaction. Likewise, inhibition of RIP in addition to the above inhibition of TRADD binding to MORT-1 may further modulate Fas-p55 TNF-receptor interaction.
Recent studies have also implicated a group of cytoplasmatic thiol proteases which are structurally related to the Caenorhabditis elegans protease CED3 and to the mammalian interleukin-1β converting enzyme (ICE) in the onset of various physiological cell death processes (reviewed in Kumar, 1995 and Henkart, 1996). There have also been some indications that protease(s) of this family may take part in the cell-cytotoxicity induced by Fas and TNF receptors. Specific peptide inhibitors of the proteases and two virus-encoded proteins that block their function, the cowpox protein crmA and the Baculovirus p35 protein, were found to provide protection to cells against this cell-cytotoxicity (Enari et al., 1995; Los et al., 1995; Tewari et al., 1995; Xue et al., 1995; Beidler et al., 1995). Rapid cleavage of certain specific cellular proteins, apparently mediated by protease(s) of the CED3/ICE family, could be demonstrated in cells shortly after stimulation of Fas or TNF receptors (both the p55-TNF receptor and the p75-TNF receptor).
One such protease and various isoforms thereof (including inhibitory ones), designated MACH which is a MORT-1 binding protein and which serves to modulate the activity of MORT-1 and hence of Fas and p55-TNF receptor, and which may also act independently of MORT-1, has been recently isolated, cloned, characterized, and its possible uses also described, as is set forth in detail in the international application No. PCT/US96/10521, and in a recent publication (Boldin et al., 1996). Another such protease and various isoforms thereof (including inhibitory ones), designated Mch4 has also recently been isolated and characterized (Fernandes-Alnemri et al., 1996; Srinivasula et al., 1996). This Mch4 protein is also a MORT-1 binding protein which serves to modulate the activity of MORT-1 and hence likely also of Fas and p55-TNF receptor and which may also act independently of MORT-1.
Moreover, it has also recently been found that besides the above noted cell cytotoxicity activities and modulation thereof mediated by the various receptors and their binding proteins including Fas, p55-TNF receptor, MORT-1, TRADD, RIP, MACH and Mch4, a number of these receptors and their binding proteins are also involved in the modulation of the activity of the nuclear transcription factor NF-κB, which is a key mediator of cell survival or viability, being responsible for the control of expression of many immune- and inflammatory-response genes. For example, it has been found that TNF-α can actually stimulate activation of NF-κB and thus TNF-α is capable of inducing two signals in cells, one eliciting cell death and another that protects cells against death induction by inducing gene expression via NF-κB (see Beg and Baltimore, 1996; Wang et al., 1996; Van Antwerp et al., 1996). A similar dual effect for Fas has also been reported (see reference to this effect as stated in above Van Antwerp et al., 1996). It would therefore appear that there exists a delicate balance between cell death and cell survival upon stimulation of various types of cells with TNF-α and/or the Fas-L, the ultimate outcome of the stimulation depending on which intracellular pathway is stimulated to a greater extent, the one leading to cell death (usually by apoptosis), or the one leading to cell survival via activation of NF-κB.
In addition, recently there has been further elucidated the possible pathway by which members of the TNF/NGF receptor family activate NF-κB (see Malinin et al., 1997 and the various relevant references set forth therein). Briefly, it arises that several members of the TNF/NGF receptor family are capable of activating NF-κB through a common adaptor protein, Traf2. A newly elucidated protein kinase called NIK (see above Malinin et al., 1997) is capable of binding to Traf2 and of stimulating NF-κB activity. In fact, it was shown (see aforesaid Malinin et al.) that expression in cells of kinase-deficient NIK mutants results in the cells being incapable of having stimulation of NF-κB in a normal endogenous manner and also in the cell having a block in induction of NF-κB activity by TNF, via either the p55-TNF receptor or Fas, and a block in NF-κB induction by TRADD, RIP and MORT-1 (which are adaptor proteins that bind these p55-TNF and/or Fas receptors). All of the receptors p55-TNF and p75-TNF receptors and Fas, and their adaptor proteins MORT-1, TRADD and RIP bind directly or indirectly to Traf2, which by its binding ability to NIK apparently modulates the induction of NF-κB.
It has been a long felt need to provide a way for modulating the cellular response to Fas-L and to TNF. For example, in the pathological situations mentioned above, where Fas-L or TNF is overexpressed or otherwise present in excess amounts or where the Fas or, at least, p55-TNF receptor is over-activated or overexpressed, it would be desirable to inhibit the Fas-L or TNF-induced cytocidal effects, which in other situations, e.g. in tumor cells or wound healing applications, it would be desirable to enhance the TNF effect, or in the case of Fas, in tumor cells or HIV-infected cells, it would be desirable to enhance the Fas-mediated effect. To this end, a number of approaches have been attempted directed at the receptors themselves (to enhance or to inhibit their activity or amount, as the case may be) or directed at the signaling pathways, as noted above, in which these receptors or their associated proteins play a role (to enhance or inhibit the activities or amounts of the receptors or their associated proteins, as the case may be).
However, heretofore there has not been elucidated the role of glucocorticoid hormones (GCH) in the regulation of lymphocyte apoptosis, in particular, the role that GCH have in inducing gene expression, the product(s) of which may modulate apoptosis in T cells (and possibly other cells as well), which modulation may be by direct or indirect interaction with, or other means of modulation of, the Fas-mediated or the associated/related p55-TNF receptor-mediated intracellular signaling pathways leading to cell death (by apoptosis in which various proteases as noted above are involved) or leading to cell survival (via induction of NF-κB activation as noted above).